Agonists and antagonists of the somatostatin receptor   

The invention relates to substituted β3-Phe-Trp-β3-Lys-beta-tri-peptides and derivatives thereof, a process for their preparation, pharmaceutical preparations which contain these compounds which are agonists/antagonists of somatostatin receptors, as active agents for the treatment of disorders which can be influenced by a modulation of somatostatin receptor activity, in particular somatostatin receptor sst4 activity, by the compounds of the invention.

Somatostatin (SRIF) is a hormone which acts with G protein-coupled receptors to influence a variety of cellular processes. It naturally occurs in two major cyclic forms: as a tetradecapeptide and as a 28-amino acid form. It is known to affect cell growth and to inhibit the secretion of hormones and neurotransmitters such as catecholamine, insulin, growth hormone, Ghrelin, glucagon, gastrin, secretin and bile, among others. These diverse biological activities of SRIF are mediated by a family of five different receptors sst1 to sst5, which SRIF binds equally strongly in the low picomolar range. However, the extent of functional redundancy between the different somatostatin receptors is not known.

Somatostatin is currently thought to play a major role in the regulation of hormone/transmitter release, both in the brain and periphery, including gut, pancreas and lung. As a result, this peptide has pleiotropic effects on whole body/systemic functions, such as growth and homeostasis, where it influences the secretion of various mediators. In the brain, for example, somatostatin regulates the hypothalamic-pituitary axis, blocking the release of growth hormone.

The following details about the molecular mechanisms by which somatostatin controls secretion are known: somatostatin is a ligand for a family of 7TM G-protein-coupled receptors, sst1 to sst5, which differ in the distribution and the pathways to which they couple. Through G-proteins, these receptors affect several pathways, including inhibiting adenylate cyclase (AC) and cAMP signaling, and activating protein tyrosine phosphatases, PLD and PLA. These receptors also influence K, Ca and Na channel function and intracellular Ca mobilisation. These mechanisms enable the inhibition of hormone secretion and effects on proliferation by somatostatin. Specifically, via G-proteins sst4 is known to inhibit cAMP signaling, active PLD and PLA2, alter Ca/H channel activity, inhibit Na/K exchanger NHI1 and activate the MAPK pathway. These pathways lead to an inhibition of exocytosis of synaptic residues and granules, including of GABA and glutamate release, and the promotion of proliferation.

Considering the pleiotropic effects of somatostatin, it is desirable to be able to selectively induce specific effects, in specific tissues if possible. While SRIF receptor subtypes have been characterized by molecular cloning and pharmacology, the availability of selective ligands for individual subtypes is still relatively limited. The first synthetic peptide analogues of SRIF, e.g. octreotide, bind with a similar affinity to two or more receptor subtypes. Recently however, Rivier et al. (2003) have developed octapeptides with a high selective affinity to the sst4 receptor. Some of these peptides have proved to be clinically useful and are indicated for the treatment of acgromegaly, pancreatic tumors and other functional gastro-intestinal disorders, for example. Most of these peptide somatostatin agonists are rather unstable in vivo due to protease degradation. Furthermore, the few side effects of sst agonists so far reported include gastro-intestinal disorders, and the occurrence of cholesterol gall stones.

Sst4 expression in rat (similar to human) occurs in the brain, gut and pancreas. It is also the sole somatostatin receptor expressed in the lung. In the brain, moderate but widespread expression is found in the cortex, where sst4 colocalises with sst2 on somatodendrites, in the hippocampus, where localization is different to and separate from sst2 and is found in the hypothalamus and the pituitary. The specific role played by sst4 in each of these organs is not known and is complicated by the presence of other ssts.

More recently, a series of non-peptide agonists, which are subtype selective and have a high receptor affinity, have been reported for each of the 5 human SRIF receptor subtypes (for a review see Weckbecker et al. 2003). When synthesising SRIF analogues, preservation of the core residues D-Trp8-Lys9 of SRIF has been thought to be an absolute prerequisite for full receptor recognition and bioactivity. Studies recently carried out by Grace et al. (2003) indicate that the backbone conformation of the peptide is not important in binding to the sst4 receptor, but forms a scaffold to orient the side chain of the essentially important residues, namely indol at position 8, amino alkyl function at position 9 and an aromatic ring in the respective positions for effective receptor ligand binding.

Liu et al (1998) describe a non-peptide somatostatin derivative, NNC 26-9100, which utilizes a novel thiourea scaffold to mimic the Trp8 residue, a non-hetero aromatic nucleus to mimic Phe7 and a primary amine or other basic probe to mimic the Lys9 residue of somatostatin, resulting in an affinity of KD=6 nM. Studies are currently in progress to evaluate the therapeutic potential for the treatment of glaucoma.

Souers et al. (2000) describe a subtype selective somatostatin mimetic prepared by incorporating conformational constraints into a nine membered heterocyclic scaffold having an affinity for the sst4 receptor up to KD=41 nM.

Using a glucose-based peptido-mimetic approach Hirschmann et al. (2003) obtained somatostatin analogues with a binding affinity of KD=53 nM and enhanced water solubility.

By molecular modelling of the somatostatin pharmacore, Rohrer et al. (1998) isolated an sst4 receptor selective compound from a combinatorial library. In binding and functional assays, L-803, 087 proved to be a hsst4 receptor agonist (KD=0.7 nM). L-803, 087 did not inhibit the secretion of growth hormone, insulin or glucagon.

Biomolecules (like peptides, nucleotides or steroids) are tolerated in the body and often show high affinities for biological target classes, but do often not fulfill criteria of oral bioavailability. In that sense, they are expected to have only low absorption and permeability, and are unattractive as candidates for drug development. Additionally, the fast proteolytic degradation of peptides based on α-amino acids resulting in a very short in vivo half life time is also a major drawback in the action of native somatostatin.

In order to overcome these problems, analogues of biomolecules, e.g. β-peptides having high affinity and selectivity for hsst4 receptors have been developed (Seebach et al., 2001, Gademann et al., 2001). These β-peptides, however, have only moderate oral bioavailability.

Thus, an object of the present invention was the provision of novel sst4 receptor binding compounds with increased bioavailability, particularly for oral administration. Surprisingly, it was found that fatty acid conjugates of mixed α/β3-tetrapeptide-based somatostatin analogues have a higher affinity for the sst4 receptor and improved pharmacologic properties, e.g. an improved bioavailability compared to known sst4 receptor agonists. The compounds of the invention have emerged as a promising new class of somatostatin agonists by combining hsst4-receptor subtype selectivity with the resistance against proteolysis.

The invention relates to compounds of the general Formula I

wherein R1=COR7 or R7, wherein R7 is a linear or branched C1-C12 alkyl group, a linear or branched C2-C12 alkenyl group, a linear or branched C2-C12 alkynyl group, or a saturated/unsaturated, aromatic or heteroaromatic mono- or polycyclic group, wherein said alkyl, alkenyl or alkynyl group may be mono- or polysubstituted with halo, hydroxy, C1-C4 alkoxy, carboxy, C1-C4 alkoxy carbonyl, amino, C1-C4 alkyl amino, di-(C1-C4-alkyl) amino, cyano, carboxy amide, carboxy-(C1-C4-alkyl) amino, carboxy-di(C1-C4-alkyl) amino, sulfo, sulfido (C1-C4-alkyl), sulfoxido (C1-C4-alkyl), sulfono (C1-C4-alkyl), thio or a saturated, unsaturated, aromatic or heteroaromatic, mono- or polycyclic group, wherein said cyclic group may be mono- or polysubstituted with halo, hydroxy, C1-C4-alkoxy, carboxy C1-C4 alkoxycarbonyl, amino, C1-C4-alkylamino, di(C1-C4-alkyl) amino, cyano, carboxy amide, carboxy (C1-C4-alkyl) amido, carboxy-di(C1-C4-alkyl) amido, sulfo, sulfido (C1-C4-alkyl), sulfoxido (C1-C4-alkyl), sulfono (C1-C4-alkyl), thio, C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl; R2 is hydrogen or C1-C4 alkyl, R3 is hydrogen or C1-C4 alkyl, which may be substituted with a saturated, unsaturated, aromatic or heteroaromatic, mono- or polycyclic group, R4 is hydrogen or C1-C4 alkyl, R5 is hydrogen or C1-C4 alkyl, and R6=(Y)n(—NR8R9)m, wherein Y is the residue of an amino carboxylic acid, particularly of a β-aminocarboxyclic acid, wherein Y may form a cyclic group; n=0 or 1, m=0 or 1, R8 and R9 are independently hydrogen, a linear or branched C1-C12 alkyl group, a linear or branched C2-C12 alkenyl group, a linear or branched C2-C12 alkenyl group, or a saturated, unsaturated, aromatic or heteroaromatic mono- or polycyclic group, wherein said alkyl, alkenyl or alkynyl group may be mono- or polysubstituted with halo, hydroxy, C1-C4 alkoxy, carboxy, C1-C4 alkoxy carbonyl, amino, C1-C4 alkyl amino, di-(C1-C4-alkyl) amino, cyano, carboxy amide, carboxy-(C1-C4-alkyl) amino, carboxy-di(C1-C4-alkyl) amino, sulfo, sulfido (C1-C4-alkyl), sulfoxido (C1-C4-alkyl), sulfono (C1-C4-alkyl), thio or a saturated, unsaturated, aromatic or heteroaromatic, mono- or polycyclic group, wherein said cyclic group may be mono- or polysubstituted with halo, hydroxy, C1-C4-alkoxy, carboxy C1-C4 alkoxycarbonyl, amino, C1-C4-alkylamino, di(C1-C4-alkyl) amino, cyano, carboxy amide, carboxy (C1-C4-alkyl) amido, carboxy-di(C1-C4-alkyl) amido, sulfo, sulfido (C1-C4-alkyl), sulfoxido (C1-C4-alkyl), sulfono (C1-C4-alkyl), thio, C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl; or wherein R8 and R9 together form a cyclic group, preferably a 5- or 6-membered cyclic group; or salts or derivatives thereof in the form of individual enantiomers, diastereomers or mixtures thereof.

Preferred are compounds of Formula I in which R7 can be either an unsubstituted or a substituted C1-C10 alkyl residue or an unsubstituted or a substituted cyclic group. Particularly preferred are methyl, ethyl, butyl, nonyl, cyclohexyl, phenyl, ethylphenyl and adamantyl.

R2 is preferably hydrogen or methyl. R3 is preferably hydrogen, methyl, phenyl or ethyl. Preferably, R4 and R5 are independently hydrogen and methyl residues. More preferably, R4 and R5 are hydrogen.

The substituent n may be 0 or 1. When n=1, Y is preferably a β-amino acid residue, wherein R8 is an unsubstituted or a substituted C1-C10, particularly C2-C8 alkyl group or an unsubstituted or a substituted cyclic group, e.g. a β-threonine residue which may form a lactone group or a β-valine residue or a β-amino acid derivative, particularly a β-amino acid amide, e.g. an optionally substituted β-threonine amide or β-valine amide.

The substituent m is preferably 1, i.e. is present, for example, as an amide group as indicated above. Preferably, at least one of R8 and R9 is an unsubstituted or a substituted C1-C10, particularly C2-C8 alkyl group or an unsubstituted or a substituted cyclic group.

R8 is more preferably ethyl, butyl, pentyl, hexyl, ethylphenyl or cyclopentyl. When R9 is other than hydrogen, it is preferably an unsubstituted C1-C2 alkyl group, e.g. methyl or ethyl.

Specific examples of the compounds of the present invention preferably include those compounds of Formula I in which R1 represents COR7 and R6 represents a β-threonine amide. These are the compounds of Formula Ia according to the present invention

wherein R7, R2, R3, R4, R5, R8 and R9 are as defined above.

Further preferred examples of the compounds of the present invention are those compounds of Formula I wherein R1=COR7 and R6 represents threonine lactone. These are the compounds of Formula 1b according to the present invention

wherein R7, R2, R3, R4, R5 are as defined above.

Preferred examples of the compounds of the present invention are those compounds of Formula I wherein R1=COR7 and R6 represents a β-valine-amide. These are the compounds of Formula Ic according to the present invention

wherein R7, R2, R3, R4, R5, R8 and R9 are defined as above.

Further preferred examples of the compounds of the present invention include those compounds of Formula I wherein R1=COR7, and R6=NR8R9. These are the compounds of Formula 1d according to the present invention

wherein R7, R2, R3, R4, R5, R8 and R9 are as defined above.

The invention also relates to the physiologically acceptable salts and derivates of the compound of Formula I.

The physiologically acceptable salts may be obtained in a conventional way by neutralizing the acids with inorganic or organic bases. Examples of suitable inorganic acids are hydrochloric acid, sulfuric acid, phosphoric acid or hydrobromic acid, and examples of suitable organic acids are carboxylic acid or sulfonic acids, such as acetic acid, tartaric acid, lactic acid, propionic acid, glycolic acid, malonic acid, maleic acid, fumaric acid, tannic acid, succinic acid, alginic acid, benzoic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, cinnamic acid, mandelic acid, citric acid, malic acid, salicylic acid, 3-aminosalicylic acid, ascorbic acid, embonic acid, nicotinic acid, isonicotinic acid, oxalic acid, amino acids, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid or naphthalene-2-sulfonic acid. Examples of suitable inorganic bases are sodium hydroxide solution, potassium hydroxide solution, ammonia and suitable organic bases are amines, but preferably tertiary amines such as trimethylamine, triethylamine, pyridine, N,N-dimethylaniline, quinoline, isoquinoline, α-picoline, β-picoline, γ-picoline, quinaldine or pyrimidine.

Physiologically acceptable salts of the compounds of Formula I can additionally be obtained by converting derivatives having tertiary amino groups in a manner known per se with quaternizing agents into the corresponding quaternary ammonium salts. Examples of suitable quaternizing agents are alkyl halides such as methyl iodide, ethyl bromide and n-propyl chloride, but also arylalkyl halides such as benzyl chloride or 2-phenylethyl bromide.

The invention also relates to derivatives of the compounds of Formula I which are preferably compounds which are converted, e.g. hydrolyzed, under physiological conditions to compounds of Formula I or into which the compounds of Formula I are metabolized under physiological conditions.

The invention further relates to optical enantiomers or diastereomers or mixtures of compounds of Formula I which contain an asymmetric carbon atom, and in the case of a plurality of asymmetric carbon atoms, also the diastereomeric forms. Compounds of Formula I which contain asymmetric carbon atoms and which usually result as racemates can be separated into the optically active isomers in a manner known per se, for example with an optically active acid. However, it is also possible to employ an optically active starting substance from the outset, in which case a corresponding optically active or diastereomeric compound is obtained as the final product.

The compounds of the invention have been found to have pharmacologically important properties which can be utilized in therapy. The compounds of Formula I can be employed alone, in combination with one another or in combination with other active ingredients.

The compounds of the present invention are β-peptide derivatives with a high affinity to human somatostatin receptors, particularly to the hsst4 receptor and high bioavailability. Preferably, the KD is ≦ about 2 μM, more preferably the KD is ≦200 nM and most preferably the KD is ≦ 50 nM. Thus, an aspect of the invention that the compounds of Formula I or the salts thereof can be used for the treatment of disorders in which a modulation of hsst4-signaling is beneficial. This modulation includes effects on the differentiated gene expression in response to the compounds of Formula I. This includes groups of genes related to the known molecular mechanism/signaling of sst4 activity, such as calcium regulators, sodium calcium and potassium channels, MAP kinases, phosphatases and cAMP signaling. Via these mechanisms, sst4 affects growth, metabolism, hormonal regulation and secretion of hormones. For instance, sst4-signaling can affect proliferation via MAPK signaling, ERK, p53 and Rb and phosphatases (Patel, 1999; Weckbecker et al. 2003). The sst4 receptor can also affect secretion via inhibition of cAMP/Ca2+-signals or via modulation of Ca/K channels on phosphotidylinositol signaling via phosphalipases. Linked to sst4 activity are also genes for neurotransmitters/hormones such as VEGF (Mentelein et al., 2001) and glutamate (Moneta et al., 2002).

Examples of disorders and diseases which can be treated by sst4 receptor agonists such as the compounds of the invention are reported in WO2005082844, which teaching is incorporated herein by reference. Disorders arising from this sst4 receptor activity include disorders of the central nervous system, in particular epilepsy, impaired behaviour such as impaired learning and memory or attention deficit disorder and pain, including chronic pain. Further possible uses are the treatment of patients suffering from neurological disorders, such as neurodegenerative diseases, in particular Alzheimer\'s disease, Parkinson\'s disease and multiple sclerosis.

The compounds of the invention can likewise be used for the treatment of hyperproliferative disorders, in particular of endocrine and solid tumors, for example for the treatment of acromegaly, melanomas, breast cancer, prostate adenomas and prostate cancer, lung cancer, bowel cancer, skin cancer and leukemias.

The compounds of the invention can be used for the treatment of diseases associated with vascular remodelling such as restenosis or the treatment of chronic transplant rejection. It can also be used for the treatment of post-surgical symptoms, such as brain aneurysms and postsurgical vascular re-stenosis. The compounds of the invention can be used for the treatment of wounds, the promotion of wound healing or tissue repair.

The compounds of the invention can be used for the treatment of gastrointestinal disorders such as diarrhoea and chemotherapy-induced and AIDS-related diarrhoea, as well as in the treatment of acute variceal bleeding. The compounds of the invention can be used for the treatment of inflammatory disorders including inflammations of the joints, including arthritis and rheumatoid arthritis, and other arthritic disorders such as rheumatoid spondylitis. Also possible is the treatment of psoriasis, Graves disease and inflammatory bowel disease.

Further possible use of the compounds of the invention are the treatment of allograft rejection. The compounds of the invention can be used for the treatment of diabetic retinopathy and nephropathy and diabetic angiopathies.

The compounds of the invention can be used in the treatment of ophthalmologic disorders, for example, age-related macula degeneration and glaucoma diabetic retinopathy. The compounds of the invention can also be used in the treatment of benign prostatic hyperplasia.

The compounds of the invention can also be labelled and used for diagnosis, e.g. radiodiagnosis and/or radiotherapy of SRIF receptor-expressing tumors, as well as the regression of otherwise unresponsive tumors.

The drug products are produced by using an effective dose of the compounds of the invention or salts thereof, in addition to conventional adjuvants, carriers and additives. The dosage of the active ingredients may vary depending on the route of administration, the age and weight of the patient, the nature and severity of the disorders to be treated and similar factors. The daily dose may be given as a single dose to be administered once a day, or divided into 2 or more daily doses, and is usually 0.001-100 mg. Daily dosages of 0.1-50 mg are particularly preferred.

Oral, parenteral, intravenous, transdermal, topical, inhalational and intranasal preparations are suitable as administration forms. Topical, inhalational and intranasal preparations of the compounds of the invention are particularly preferred. Galenical pharmaceutical presentations such as tablets, coated tablets, capsules, dispersible powders, granules, aqueous solutions, aqueous or oily suspensions, syrup, solutions or drops are used.

Solid drug forms may comprise inert ingredients and carriers such as, for example, calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatin, guar gum, magnesium stearate or aluminium stearate, methylcellulose, talc, colloidal silicas, silicone oil, high molecular weight fatty acids (such as stearic acid), agar-agar or vegetable or animal fats and oils, solid high molecular weight polymers (such as polyethylene glycol); preparations suitable for oral administration may, if desired, comprise additional flavourings and/or sweetners.

Liquid drug forms can be sterilized and/or, where appropriate, can comprise excipients such as preservatives, stabilizers, wetting agents, penetrants, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols to control the osmotic pressure or for buffering and/or viscosity regulators.

Examples of such additions are tartrate buffer and citrate buffer, ethanol, complexing agents (such as ethylenediaminetetraacetic acid and its non-toxic salts). Suitable for controlling the viscosity are high molecular weight polymers such as, for example, liquid polyethylene oxide, microcrystalline celluloses, carboxymethylcelluloses, polyvinylpyrrolidones, dextrans or gelatin. Examples of solid carriers are starch, lactose, mannitol, methylcellulose, talc, colloidal silicas, higher molecular weight fatty acids (such as stearic acid), gelatin, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable fats, solid high molecular weight polymers such as polyethylene glycol.

Oily suspensions for parenteral or topical uses may be vegetable, synthetic or semisynthetic oils such as, for example, liquid fatty acid esters with, in each case, 8 to 22 C atoms in the fatty acid chains, for example palmitic, lauric, tridecyclic, margaric, stearic, arachic, myristic, behenic, pentadecyclic, linoleic, elaidic, brasidic, erucic or oleic acid, which are esterified with monohydric to trihydric alcohols having 1 to 6 C atoms, such as, for example, methanol, ethanol, propanol, butanol, pentanol or isomers thereof, glycol or glycerol. Examples of such fatty acid esters are commercially available miglyols, isopropyl myristate, isopropyl palmitate, isopropyl stearate, PEG 6-capric acid, caprylic/capric esters of saturated fatty alcohols, polyoxyethylene glycerol trioleates, ethyl oleate, waxy fatty acid esters such as artificial duch preen gland fat, coco fatty acid, isopropyl ester, oleyl oleate, decyl oleate, ethyl lactate, dibutyl phthalate, diisopropyl adipate, polyol fatty acid esters inter alia. Also suitable are silicone oils differing in viscosity or fatty alcohols such as isotridecyl alcohol, 2-octyldodecanol, cetylstearyl alcohol or oleyl alcohol, fatty acids such as, for example, oleic acid. It is also possible to use vegetable oils such as caster oil, almond oil, olive oil, sesame oil, cottonseed oil, peanut oil or soybean oil.

Suitable solvents, gel formers and solubilizers are water or water-miscible solvents. Suitable examples are alcohols such as, for example, ethanol or isopropyl alcohol, benzyl alcohol, 2-octyldodecanol, polyethylene glycols, phthalates, adipates, propylene glycol, glycerol, di- or tripropylene glycol, waxes, methyl Cellosolve, Cellosolve, esters, morpholines, dioxane, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, cyclohexanine, etc.

Film formers which can be used are cellulose ethers able to dissolve or swell both in water and in organic solvents such as, for example, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose or soluble starches.

Combined forms of gel formers and film formers are also possible. In particular, ionic macromolecules are used for this purpose, such as, for example, sodium carboxymethylcellulose, polyacrylic acid, polymethylacrylic acid and salts thereof, sodium amylopectin semiglycolate, alginic acid or propylene glycol alginate as sodium salt, gum arabic, xanthan gum, guar gum or carrageenan.

Further formulation aids which can be employed are glycerol, paraffin of differing viscosity, triethanolamine, collagen, allantoin, novantisolic acid.

It may also be necessary to use surfactants, emulsifiers or wetting agents for the formulation, such as, for example, Na lauryl sulfate, fatty alcohol ether sulfates, di-Na—N-lauryl-β-iminodipropionate, polyethoxylated castor oil or sorbitan monooelate, sorbitan monostearate, polysorbates (e.g. Tween), cetyl alcohol, lecithin, glyceryl monostearate, polyoxyethylene stearate, alkylphenol polyglycol ether, cetyltrimethylammonium chloride or mono/dialkylpolyglycol ether orthophosphoric acid monoethanolamine salts.

Stabilizers such as montmorillonites or colloidal silicas to stabilize emulsions or to prevent degradation of the active substances, such as antioxidants, for example tocopherols or butylated hydroxyanisole, or preservatives such as p-hydroxybenzoic esters, may likewise be necessary where appropriate to prepare the desired formulations.

Preparations for parenteral administration may be present in separate dose unit forms such as, for example, ampoules or vials. Solutions of the active ingredient are preferably used, preferably aqueous solutions and especially isotonic solutions, but also suspensions. These injection forms can be made available as a finished product or be prepared only immediately before use by mixing the active compound, e.g. the lyophilistate, where appropriate with further solid carriers, with the desired solvent or suspending agent.

Intranasal preparations may be in the form of aqueous or oily solutions or of aqueous or oily suspensions. They may also be in the form of lyophilistates which are prepared before use with the suitable solvent or suspending agent.

The manufacture, bottling and closure of the products takes place under the usual antimicrobial and aseptic conditions.

The invention further relates to a process for the manufacture of the compounds of the invention (FIG. 1).

According to the present invention, the compounds of general Formula I are manufactured according to the definitions for R1, R2, R3, R4, R5, R6, R7, R8 and R9 as previously given such that the synthetic protocol involves three efficient peptide coupling steps employing the same chemical reagents and three Boc-cleavage reactions using HCl in 1,4-dioxane. As the five-ring lactone demonstrates to be very stable against ring opening even when treated with strong carboxylic acid activating agents, the synthon can be used in all peptide coupling steps without utilization of protecting groups. With the growing peptide chain, solubility becomes a major concern. The final N-Boc-protected mixed α/β3-tetrapeptide proves to be potentially insoluble in lots of standard solvents used in peptide chemistry. The restricted, but partial solubility of the scaffold molecule in dichloromethane is sufficient to purify intermediate compounds by liquid/liquid extraction. Purification is finally achieved by extraction under weak acidic conditions established with aqueous citric acid, in order to prevent partitioning of the fully protonated product molecule (a weak base) between aqueous and organic phase.

After N-terminal-derivatization of the mixed (α/β3)-tetrapeptide scaffold with fatty acid analogues in parallel synthesis mode, deprotection of the Cbz-protecting group was achieved by hydrogenolysis (Pd on activated charcoal) in DMA under acidic conditions. Addition of trifluoroacetic acid to the solvent led to an acceleration of the hydrogenation process. In addition, immediate protonation of the so-generated primary amine inhibited a (possible) nucleophilic attack on the adjacent C-terminal five-ring-lactone. Thus, the formation of a macrocyclic lactam could be prevented. The final products were then purified by RP-chromatography leading to purities >95% as determined by HPLC, HR-MS, MS, LC-MS, 1D- and 2D-NMR spectroscopy.

The C-terminal five-ring lactones can be exchanged for their corresponding open-chain amide analogues. This was achieved by reacting the fatty acid derivatized-(α/β3)-tetrapeptides with ammonia in methanol. Due to the folding and unique structural properties of these β-amino acid containing tetrapeptides, initial reaction times range from 24 hours (nonanoyl-derivative, compounds 16 and 17 in Table 1) to 36 days (cyclohexyl-derivative, compound 26). Nonetheless, the reaction times can be accelerated by dissolving the lactone containing tetrapeptides in N,N-dimethylacetamide (DMA) and subsequent addition of ammonia in methanol. Conversion rates are generally near hundred percent (>98%) and due to the high purity (>95% as determined by RP-HPLC) of the generated C-terminal amides, further purification was not necessary.

In subsequent peptide series, primary or secondary amine building blocks are introduced into the peptide by reaction of the fully protected C-terminal β3-amino acids (Nα-Boc-Nω-Z-(S)-β3-HLys and Boc-(R)-β3-Leucine) employing carbonyldiimidazole activation chemistry, followed by deprotection and subsequent coupling.

Double conjugated biomolecules (Formula Id, R6=NR8R9) consisting of only three amino acids (two β3 and one α) show much better solubility in organic solvents and lead to an acceleration in work up procedures by avoiding hardly separable emulsions. The same is observed for beta-peptides when capped with N-alkylated groups in the amide backbone.

The generated peptides are tested for their affinity to bind to human SRIF receptors expressed in Chinese hamster lung fibroblast (CCL39) cells. This is achieved in radioligand-binding assays, a displacement experiment in which the concentration of a substance is measured which is necessary for the replacement of 50% of a specifically bound radioligand ([125I]LTT-SRIF28). Specific binding is measured as the total binding of receptor-specific radioligand minus the amount of radioligand bound in presence of unmarked SRIF-14 (100 nM, nonspecific binding).

TABLE 1 List of all tested compounds based on scaffold I with the corresponding compound numbers. N-terminal R1 Building Blocks R2 C-terminal H 1, 2, 3 16, 17 26 18 14 15 35, 36 7, 8, 9 39 (R3 = Me) 21 (R4 = Me) 11, 12 5 13 20 (R4 = Me) 6 10 19 (R4 = Me) 28, 29 38 (R3 = Me)

Download full PDF for full patent description/claims.




You can also Monitor Keywords and Search for tracking patents relating to this Agonists and antagonists of the somatostatin receptor patent application.
###


Other recent patent applications listed under the agent :